Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/722—Chitin, chitosan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/734—Alginic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/26—Mixtures of macromolecular compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/52—Hydrogels or hydrocolloids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P23/00—Anaesthetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus

Definitions

• the present invention relates to a method of producing spherical hydrogel beads comprising a mixture of chitosan and alginate.
• the present invention also relates to a new supplementation method and composition for treating joint disorders, particularly osteoarthritis.
• Osteoarthritis is a progressive degenerative disorder accompanied by pain and characterized by a breakdown of cartilage in articular joints, a deterioration of the synovial fluid present in the articular joints, and a resulting
• Osteoarthritis increases with age, with a probability higher than 60% of those 60 years old or older.
• the present therapy is generally, either therapeutic with administration of analgesic or antiinflammatory agents or a surgical therapy with partial or total joint replacement.
• An alternative approach is viscosupplementation which is an injection into the joint of a biocompatible viscous (e.g. Hyaluronic acid) lubricant that reduces friction and pain. Viscosupplementation is used to supplement synovial fluid that lubricates and protects the articular joints. Indeed, in OA patient, the synovial fluid is modified with a decrease of concentration and molecular weight (Mw) of hyaluronan (or salt of hyaluronic acid HA) .
• Mw concentration and molecular weight
• Intra-articular injections comprising high molecular weight of HA preparations are currently available and used to treat knee, hip, carpometacarpal joint of the thumb or ankle. These preparations require one (Durolane TM, Q-Med AB, Uppsala, Sweden), three (Synvisc ® , high molecular weight cross-linked HA, Orthovisc ® ) to five (Hyalgan ® , Supartz ® ) intra-articular (IA) injections (1 to 3 ml containing 5 to 20 mg/ml HA per injection according the joint).
• IA intra-articular
• HA preparations vary in a number of characteristics, including for example, the source of HA (animal-derived or bacterial), the concentration and Mw of HA and the type and degree of chemical crosslinking used, if any. Usually, most injectable HA preparations, once injected, have residence half-life between hours to several days. Several studies have compared the efficacy of the above-mentioned products and differently concluded to a marked reduction of pain and improved function of patients with knee OA. Some concluded that at best there is small effect compared to placebo injection.
• WO2007/1351 14 describes viscosupplementation with polysaccharides compositions of a mixture of alginate and chitosan without generating insoluble coacervates. Coacervates are due to the polycationic nature of chitosan that makes compatibility difficult with other polysaccharides such as hyaluronic acid (a polyanion) or alginate. According to WO2007/1351 14, precipitation/coarcervation of polysaccharides such as chitosan and alginate prevents any formulation thereof as an injectable composition.
• WO2007/1351 14 a composition with an aqueous mixture of chitosan of a high degree of derivatization of at least 40% and an alginate is therefore described as highly viscous without generating insoluble coacervates.
• One aspect of the present invention relates to a method of producing a hydrogel matrix comprising the steps of:
• the droplets of the mixed solution are introduced in a solution of Sr 2+ ions.
• the mixed solution comprises 0.6 % chitosan and or comprises 1 .2 % alginate.
• the ratio between alginate and chitosan in the mixed solution is between 1 .4 and 2.8, or between 1 .75 and 2.25 or is about 2.
• the chitosan has a Mw of between 35 and 45 kD and/or is of animal or preferably of vegetable origin.
• the method further comprises the step of mixing the beads in a thermosensitive hydrogel.
• the ratio between beads and hydrogel is for example between 5/1 and 1/1 , or between 4/1 and 2/1 .
• the formation of beads is performed by passing droplets through a needle to obtain beads with a diameter between 0.01 and 5 mm.
• Another aspect of the present invention refers to a spherical hydrogel bead comprising a homogeneous mixture of chitosan and alginate, wherein said bead is obtainable by the methods as described above.
• Another aspect of the present invention refers to a spherical hydrogel bead with a diameter between 0.01 and 5 mm, comprising a homogeneous mixture of chitosan and alginate , characterised in that the bead comprises between 1 and 1 .4 % alginate and between 0.5 and 0.7 % chitosan, for example, the bead comprises 1 .2 % alginate, or for example the bead comprises 0.6% chitosan.
• the ratio of alginate/chitosan is between 1 .4 and 2.8, preferably between 1 .75 and 2.25, most preferably between 1 .8 and 2.2.
• the chitosan has a Mw between 35 and 45 kD and/or is of animal or vegetable origin.
• the invention also relates to an intra-articular supplement comprising spherical hydrogel beads with a diameter between 0.01 and 5 mm, comprising a homogeneous mixture of alginate and chitosan of Mw lower than 60 kD , characterised in that said beads comprise between 1 and 1 .4 % alginate and between 0.5 and 0.7 % chitosan.
• the intra-articular supplement may further comprise a viscous thermogelling gel as for example a polysaccharide hydrogel, particularly a chitosan hydrogel.
• polysaccharides By polysaccharides, one means biopolymers such as hyaluronic acid known as having repeating disaccharide units composed of glucuronic and N-acetyl- glucosamine, alginates and chitosan.
• Chitosan is a polysaccharide widely available in nature obtained by chemical deacetylation of chitin and the principal constituent of crustacean exoskeletons but also from the mushrooms wall. It is composed of D-glucosamine units and N-acetyl-D-glucosamine units.
• the intra-articular supplement may also comprise a component selected from the group consisting of non-steroidal anti-inflammatory drug, anaesthetic, opioid analgesics, corticosteroids, antineoplastic, monoclonal antibodies, chimeric monoclonal antibodies, vitamins, minerals, nutraceuticals.
• the intra-articular supplement may also contain additional active or inactive components including for example, the non-steroidal anti-inflammatory drugs (NSAIDS) e.g diclofenacTM, ibuprofenTM, piroxicamTM; anesthetics, e.g.
• NSAIDS non-steroidal anti-inflammatory drugs
• diclofenacTM e.g diclofenacTM, ibuprofenTM, piroxicamTM
• anesthetics e.g.
• LidocaineTM and BupivacaineTM LidocaineTM and BupivacaineTM; opiod analgesics, e.g codeine and morphine; corticosteroids, e.g., dexamethasone and prednisone; antineoplastic agents such as MethotrexateTM; anti-viral agents, e.g. AcyclovirTM and VidarabineTM; monoclonal antibodies e.g.HumiraTM and chimeric monoclonal antibodies e.g infliximabTM.
• Intra-articular injectable supplements may also contain
• the invention also relates to a supplementation method for treating joint disorder of a subject, and for reducing pain and discomfort associated with joint injury or joint diseases including osteoarthritis and traumatic cartilage lesions.
• joint diseases include osteoarthritis (primary (idiopathic) or secondary), rheumatoid arthritis, joint injury (e.g.traumatic or repetitive motion injury), cartilage pathology (e.g. chondrocalconsis, chondromalacia), septic arthritis.
• the invention further provides methods of reducing pain associated with such diseases, to repair bone and cartilage lesion and to slow down disease progression.
• the supplementation method of treating a joint disorder comprises
• the supplementation method may comprise a single or multiple intra-articular injections or implantation of the supplement in an amount sufficient to provide a therapeutic effect.
• the intra-articular injection or implantation is done directly in bone and/or cartilage defect of human or non-human mammals for example by arthroscopy or with an injection device such as a syringe.
• administration sites include the knee, shoulder, ankle, temporo- madibular and carpo-metacarpal joints, elbow, hip, wrist, intervertebral disc.
• the invention further provides a supplementation device comprising a pre-filled, single use syringe having a single dose of the supplements.
• Figure 1 shows beads obtained according to the method of the invention
• Figure 2 shows a section of an alginate bead (A) and a chitosan/alginate bead (B) [chitosan: dark grey trabeculae; alginate: light grey background].
• Figure 3 shows at low magnification chitosan/alginate beads embedded in a chitosan hydrogel.
• Figure 4 shows the implantation of a thermosensitive chitosan hydrogel comprising 0.6 % chitosan/1 .2 %alginate beads (A. bisporus chitosan (40 kD))
• Figure 5 shows the histological evaluation of the implant 15 days after implantation.
• A Subchondral Bone
• B Cell-colonized hydrogel
• C cell colonized Chitosan/alginate bead
• D Cell embedded in chitosan lacunae.
• One aspect of the present invention relates to a method of producing a hydrogel matrix. This method comprises the following steps:
• the hydrogel which is obtained by this method results in a homogeneous matrix of calcium alginate and chitosan.
• the matrix as obtained in the present invention differs from prior art matrices which have a core of one component, coated with a layer of another component.
• the matrix as obtained in the present invention has the advantage that the porosity of the matrix can be more accurately defined compared to matrices which are first lyophilised to obtain a certain degree of porosity.
• the matrix as obtained in the present invention has the advantage to be composed with a low molecular weight chitosan particularly between 15 and 50 kDA which spontaneously forms an homogenous network in the alginate matrix.
• the alginate and chitosan which are used to prepare the hydrogels are dissolved in strong alkaline or acidic buffers which have a sterilising effect .
• the alginate and chitosan solutions can be mixed to obtain beads with different concentrations.
• concentration of chitosan ranges from 0.5 to 0.7 %, or from 0.55 to 0.65 %.
• concentration of alginate ranges from 1 . to 1 . 4 % or from 1 . 25 to 1 . 35 %.
• a particular embodiment of hydrogel comprises about 0.6 % chitosan and about 1 .2 % alginate.
• compositions of the present invention relate to hydrogel composition and beads obtained thereof wherein the ratio between alginate and chitosan in the mixed solution is between 1 .4 and 2.8, more particularly between 1 .5 and 2.7, more particularly between 1 .6 and 2.6, or between 1 .75 and 2.25. Particular values of this ratio are about 1 .9, 1 .95, 2.0, 2.05 and 1 .
• the average size of the beads can be adapted and empirically determined by adjusting the diameter of the needle which is used to form the droplets which are introduced into the calcium or strontium solution. Envisaged herein are beads with a diameter between 0.01 and 5 mm. These dimensions provide a compromise between the ease of manipulation and the diffusion of nutrients into the beads.
• the chitosan from the present invention may be isolated from different animal sources such as crustaceans (prawn shells) or squids.
• the chitosan may be of vegetal, more of particularly fungal origin, such as Mucoralean strains, Mucor racemosus and Cunninghamella elegans, Gongronella butleri, Aspergillus niger, Rhizopus oryzae, Lentinus edodes, Pleurotus sajo-caju, Zygosaccharomyces rouxii, Candida albicans or Agaricus bisporus. Chitosan further exists in diverse types of molecular weight.
• the chain length of chitosan may contribute to the three dimensional structure of the hydrogels.
• Typical chitosans, for use in the present invention can have an average molecular weight between 15 and 50 kD, more particularly between 35 and 45 kD.
• supplements are formulated as a biphasic supplement material formed by beads and a viscous gel.
• This supplement includes a polymeric matrix ("gel”), and the spherical three-dimensional beads comprising chitosan and alginate .
• thermosensitive gel This allows to formulate an implantable or injectable gel which upon implantation, ensures an optimal space distribution in the host tissue or organ.
• Exa m pl es of thermosensitive hydrogels include poly(N-isopropylacrylamide (PNIPAAm). A particular type hereof is chitosan.
• the chitosan network within the microbeads confers particular mechanical properties at the beads such that they are less compressible and more resistant to pressure than the beads made of alginate only.
• the matrices of the present invention provide inter-connected chitosan trabeculae within an alginate gel, resulting in an environment which is favourable for cellular culture by providing an aqueous medium with a neutral pH.
• Such trabeculae are obtained by insoluble chitosan which forms coacervates which create a basket-like structure network or trabeculae when mixed with alginate.
• the trabeculae are of varying thickness and length and provide to the beads particular biological and mechanical properties such as phenotype stabilisation, deformability, elasticity and compressive modulus.
• Beads are prepared from a homogeneous mixture of chitosan (0.6 % final) and alginate (1 .2 % final). The two solutions are prepared separately before being mixed.
• the solutions of alginate and chitosan are prepared in the following way: An alginate solution 2.4 % (W/v) in 0.16 M NaOH and a solution of chitosan 1 .333 % (w/v) in 1 .666 M HAc are prepared. To 10 volumes of the alginate solution, 1 volume Of a 1 M Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) solution is added.
• the beads are mixed with a vegetable (Agaricus bisporus) chitosan hydrogel (Kitozyme, Alleur, Belgium] . Th is step is performed below 27°C to avoid hydrogel gelification. A ratio of beads/hydrogel of 3/1 (v/v) has been used.
• Example 3 Implantation in an animal model A gel as described under example 2, has been implanted in a rabbit with subchondral bone and cartilage defect joint . ( Figure 4).
• the implant was evaluated ( Figure 5 ).
• the lesion remains filled with the implant. Beads and viscous hydrogel remain constant at least two weeks after injection in the injured joint, while maintaining a safety profile.
• the implant is colonised with cells originating from the underlying bone marrow.
• Cells were encountered in the settled thermosensitive chitosan hydrogel (B) as well as in the chitosan alginate beads (C) (chitosan trabeculae are indicated by D).
• B thermosensitive chitosan hydrogel
• C chitosan alginate beads
• Example 4 Effect of chitosan molecular weight in beads formation. Different molecular weights of native chitosan have been used in the process according to the invention. Different physical parameters such as pH and viscosity of the mixed solution (with chondrocytes were measured., Osmolarity of the resulting hydrogel is measured according to a well-known technique in the art.
• Example 5 A supplementation method for treating an articular joint of a human subject or an animal with a needle.
• a needle has been used to deliver the intra-articularly supplement.
• supplement is delivered in the synovial cavity at a density of 1 ,000 to 20,000 beads of 0.1 to 0.5 ⁇ per ml in a volume of approximately 1 -2 ml per injection.
• 1 ml of physiological liquid containing 10,000 beads of 0,2 ⁇ is injected into a knee joint using a fine (e.g. 14-22 gauge, preferably 18-22 gauges) needle.
• Example 6 A supplementation method for treating an articular joint of a human subject or an animal with a trochar
• a trochar has been used to deliver the supplement in the small to medium cartilage defect (less than 1 cm 2 ).
• supplement is delivered in the cartilage through the trochar at a density of 5 to 50 beads of 1 to 5 mm diameter.
• 4 beads of 1 mm diameter are implanted in a traumatic cartilage lesion of 0.5 cm diameter and 1 mm depth diameter.

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